From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy
Figure 6
CLEM and tomography reconstruction.
COS-1 cells transfected with GFP-BIN1 were imaged by confocal microscopy, fixed by high pressure freezing and processed to 200 nm thick sections. A tomogram was reconstructed from 139 tilted images (from −69 to 69°) and manually segmented to highlight the fluorescent tubules observed previously by confocal microscopy. (A) Representative confocal z-stack projection of a transfected cell, showing GFP-BIN1 tubules. Insert: magnified view of a tubule adjacent to the nucleus. (B) Corresponding transmission-EM picture. (C and D) Higher magnifications of the area selected in (B) showing a bundle of tubules (arrowhead) passing next to the cell nucleus, in the same region highlighted in (A). (E) Top view of the 3D model depicting membrane tubules in yellow, mitochondria, nuclear envelope, endosomal vesicles and lysosomes in grey, microtubules in red and actin filament in black. A 3D video of the tomogram is available (Video S8, online). L: lysosome; M: mitochondria; N: nucleus.